"Immunoglobulins" ("Ig") are antibodies which consist of a pair of two "heavy" chains linked to a pair of two identical "light" chains; the hypothetical structure of the immunoglobulin is in the shape of a "Y" with the heavy chains forming the base of the Y, and the light chains forming the two branches. The heavy chains and light chains are separately synthesized by the immune system; such synthesis is synchronized by the immune system, such that under normal circumstances, complete immunoglobulins are produced.
Immunoglobulins are important to humans with respect to the mediation of immunity; i.e., the chains of immunoglobulins comprise antigen binding sites such that when a foreign antigen is introduced into a human host, activated B-cells synthesize Igs with specificity for the antigen. The immunoglobulins are then capable of specifically binding to the invading antigen, whereby the host can effectively mediate the removal thereof from the body.
There are two types of light chains, referred to as "kappa" and "lambda". There are several types of heavy chains and the classification of the immunoglobulins are predicated upon the heavy chain type. I.e., immunoglobulins comprising gamma (".gamma.") heavy chains are designated as "IgG"; alpha (".alpha."), IgA; mu (".mu."), IgM; delta (.delta.), IgD; and epsilon (.epsilon.) IgE.
Immunoglobulins are proteins; thus, they are comprised of amino acid sequences. Within the specific isotype of immunoglobulin, the amino acid sequences for the light chains are substantially identical, and the amino acid sequences for the heavy chains are substantially identical. Thus, using IgG as an example, approximately one-half of the light chains and three-fourths of the heavy chains have amino acid sequences that are identical from one IgG molecule to the next. The region of identical amino acid sequences is referred to as the "constant region". The remaining one-half of the IgG light chain and one-fourth of the heavy chain are composed of highly variable amino acid sequences, referred to as the "variable region". The variable regions are important in that these give rise to "antigen binding sites" i.e. the regions that bind with specificity to particular antigens. Thus, changing the amino acid sequence in a variable region produces immunoglobulins with different antigen binding sites.
This capability is of critical importance to the immune system, and hence survival, of, e.g., mammals, including humans. Antigens typically have several different "epitopes" i.e., regions to which antibodies can bind. Thus, an immunoglobulin that has a variable region specific for one epitope on the antigen will typically be unable to bind to a different epitope on that antigen; therefore, the immune system, when stimulated, will produce a variety of immunoglobulins that have different variable regions which are specific for different antigen epitopes. This is referred to as a "polyclonal immune response," i.e., a variety of immunoglobulins are secreted by the activated B-cells in response to antigenic stimulation.
The immune system is regulated such that upon stimulation, the B-cells will produce more than a sufficient amount of immunoglobulins to neutralize the invading antigen; thereafter, B-cell production of the immunoglobulins ceases in that the need for the immunoglobulins secreted in response to the antigen is negated or dissipated.
Occasionally, single, unregulated B-cell clones will continue to produce immunoglobulin of the same idiotype (i.e., identical in terms of antigen binding site). This results in at least two problems which impact on the immune system of the host: first, the proliferation and subsequent accumulation of such immunoglobulins can stimulate the production of antibodies directed thereto (which can be referred to as an "autoimmune response"); and second, the immune system is severally "strained" by the need to attack the accumulating immunoglobulins such that the ability to fight invading antigens is weakened. Immunoglobulins of the same idiotype produced by single, unregulated B-cell clones are referred to as "monoclonal gammopathies". Monoclonal gammopathies are of principal importance with respect to clinical disorders.
Monoclonal gammopathies do not necessarily cause clinical disorders in an individual. Such a situation can be referred to as "benign monoclonal gammopathy" or "monoclonal gammopathy of undetermined significance". However, many clinical disorders are associated with monoclonal gammopathy. For example, monoclonal IgM (i.e. an increase in the production of an IgM idiotype by unregulated B-cell clones) is associated with the disease Waldenstrom's macroglobulinemia. Because IgM has a (relatively) high molecular weight, an increase production thereof is associated with an increase in the viscosity of the macroglobulinemia patient's blood, referred to as "hyperviscosity". Hyperviscosity is associated with, e.g., headache, dizziness and vertigo.
Multiple myeloma is associated with an increase in IgG, IgA, IgD or IgE idiotypes, as well as kappa or lambda light chains, or gamma, alpha, mu or delta heavy chains. A major pathologic feature of multiple myeloma is bone destruction, i.e., bone deformity or acute, painful pathological fractures. Clinically, the patient may experience bone pain, infections due to decreased production of normal Ig's, and anemia. Twenty percent of myeloma patients evidence Bence Jones protein, which is a free monoclonal light chain; these light chains, because of their (relative) small size are typically excreted, and hence present, in patient urine. Multiple myeloma can also impact neural tissue (i.e., the spinal cord, nerve roots and cranial or peripheral nerves).
As is apparent, information regarding monoclonal gammopathies is of clinical value and importance to the understanding of a variety of severe and debilitating disease states. It is therefore essential that procedures be available for the identification of monoclonal gammopathies (hereinafter "MG"). Two well-known procedures for the analysis of MG are Immunoelectrophoresis ("IEP") and Immunofixation Electrophoresis ("IFE"). Both procedures are more similar than dissimilar in protocol, although interpretation of IFE results is somewhat easier compared to IEP.
IFE is a two stage procedure using agarose gel protein electrophoresis in the first stage and immunoprecipitation in the second. In a clinical setting for the determination of MG, a clinical sample (e.g., whole blood, serum, plasma, urine, cerebro spinal fluid) is placed in multiple positions ("lanes") on an agarose gel. Because immunoglobulins are proteins, they have a charge distribution such that when an electric field is applied to the gel-containing sample, immunoglobulins will traverse the gel from anionic to cationic electrode Thereafter, antisera comprising antibodies to specific immunoglobulin classes (typically IgG, IgA, IgM, kappa and lambda) are applied to specific lanes. The gel and antisera are incubated, during which time immune complexes between specific immunoglobulins and the antibodies thereto are formed. Staining solutions are then utilized to indicate the location of such immune complexes--if no MGs are present, a somewhat consistent color stain will be evidenced; if MGs are present, these will accumulate in specific regions on the gel (due to their inherent identical weight and charge) such that a color band will appear. By utilizing a reference pattern on the gel, one can then determine the MG type present on the gel. FIG. 1 provides a patient sample evidencing an IgG (lambda) monoclonal protein as identified by IFE (each of the designated headings refer to the particular antiserum applied to that lane).
The PARAGON.RTM. electrophoresis system is a commercially available system for conducting both IFE and IEP. PARAGON.RTM. is a registered trademark of Beckman Instruments, Inc., Fullerton, Calif., U.S.A. See also, U.S. Pat. No. 4,669,363 which is incorporated herein by reference.
IFE can be considered a "positive indicator" test. I.e., the presence of a particular band is indicative of the presence of an MG corresponding to a particular immunoglobulin type. A technique related to IFE which can be considered a "negative indicator" test is referred to as Immunosubtraction Electrophoresis ("ISE"). ISE, in essence, reduces by one the number of steps of IFE. In ISE, the sample can be mixed with, e.g., an insolubilized antibody directed to an immunoglobulin; if present, that immunoglobulin will bind to the insolubilized antibody and is thus "removed" from the sample. Therefore, the sample is applied to a gel, subjected to electrophoresis, and a coloring stain is applied. In a "normal" sample, the stain should be relatively consistent across the gel; in an "abnormal" sample, i.e., one that includes MG, the stain will be absent from a region on the gel, owing to the removal of that monoclonal immunoglobulin in the initial step. Thus, the absence of a particular band is indicative of the presence of the corresponding MG from the sample, hence the "negative indicator" designation.
IEP, IFE and ISE all require the use of a separation gel as well as signal-generating stains. These procedures all involve multiple steps. Thus, the procedures can be somewhat labor intensive, which can decrease throughput, an obvious impediment in a clinical setting. Additionally, there may be a concern with the amount of disposable end-products associated with these procedures--each sample requires a gel which must be properly disposed, particularly when the analysis involves clinical samples. Such a disposal scenario can further increase the allied costs associated with these procedures.
As noted, immunoglobulins are proteins which can be separated from each other using gels subjected to an electric field. Proteins, including those from clinical samples, can also be analyzed using capillary zone electrophoresis ("CZE"). See, for example, Chen, Fu-Tai A., et al. "Capillary Electrophoresis--A New Clinical Tool." Clin. Chem. 77/1:14-19 (1991); see also, U.S. Pat. No. 5,120,413. Both of these documents are incorporated herein by reference.
Capillary zone electrophoresis is a technique which permits rapid and efficient separations of charged substances. Separation of the constituents of clinical samples can typically be accomplished in less than 20 minutes, typically in less than 10 minutes. In general, CZE involves introduction of a sample into a capillary tube, i.e. a tube having an internal diameter of from about 2 to about 2000 microns (".mu.m"), and the application of an electric field to the tube. The electric potential of the field both pulls the sample through the tube and separates it into its constituent parts. I.e., each of the sample constituents has its own individual electrophoretic mobility; those having greater mobility travel through the capillary tube faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones.
CZE can be generally separated into two categories based upon the contents of the capillary columns. In "gel" CZE, the capillary tube is filled with a suitable gel, e.g., polyacrylamide gel. Separation of the constituents in the sample is predicated in part by the size and charge of the constituents travelling through the gel matrix. Gel CZE has several disadvantages, notably, the unpredictability of the gel material. I.e., such gels eventually "breakdown" or can only be used for limited analytical runs. Such unpredictability is unacceptable in any setting where numerous analytical runs are conducted.
In "open" CZE, the capillary tube is filled with an electrically conductive buffer solution. Upon ionization of the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution (i.e., the buffer solution and the sample being analyzed), must also flow in this direction to maintain electroneutrality. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. The buffer in open CZE is as stable against conduction and diffusion as the gels utilized in gel CZE. Accordingly, separations can be obtained in open CZE quite similar to those obtained in gel-based electrophoresis.
Fused silica is principally utilized as the material for the capillary tube because it can withstand the relatively high voltage used in CZE, and because the inner walls ionize to create the negative charge which causes the desired electroendosmatic flow. However, alumina, beryllium, Teflon.RTM.-coated materials, glass, quartz and conbinations of these (with or without fused silica) can also be utilized. The capillary column is typically capable of withstanding a wide range of applied electrophoretic fields of between about 10 volts per centimeter ("v/cm") up to about 1000 v/cm. The capillary column may be coated on the outside (using, e.g., a polyimide material) for ease of handling. The inner wall of the capillary may be untreated or coated with a material capable of, inter alia, reducing adsorption to the inner wall during electroendosmatic flow of the bulk solution. However, it is typically preferred that the inner wall be uncoated because typical coatings have a tendency to breakdown in an unpredictable manner. In U.S. Pat. No. 5,120,413, analysis of clinical samples was conducted using untreated capillary columns.
The results of CZE analysis are typically presented as "electropherograms", i.e., peaks of various widths and heights which correspond to the constituent parts of the sample. For example, a constituent which is present in a sample in a high concentration may evidence a peak having a large height and wide width compared to a constituent present in a (relative) low concentration. Typically, the electropherogram is derived by plotting detection units (typically ultraviolet light absorbance) on the vertical axis, and time of constituent traversal through the column to a detection region on the horizontal axis. Results can also be derived in terms of a unit value, typically derived from the areas bounded by the individual peaks.
Open CZE has many desirable qualities for, e.g., clinical sample analysis: because the analysis does not involve a gel-filled column, the inherent limitations on the number of analytical runs that can be conducted with any particular gel-filled column are avoided; when the capillary column is untreated, the aura of unpredictability which can be associated with coated columns is avoided; the sample size is small (usually on the order of 5 to 200 .mu.l of diluted sample); sample analysis time is fast, i.e. less than about 20 minutes; and the protocol lends itself to automation, thus decreasing the labor skills necessary for efficient and effective sample analysis.
What is needed, then, is a technique applicable to the analysis of monoclonal gammopathies which can provide results with a minimum of processing steps; which is easy to utilize; which provides for high throughput; and which avoids the end-product disposal problem occasioned by the use of separating gels.